New solid support for the synthesis of 3′-oligonucleotide conjugates through glyoxylic oxime bond formation

Article abstract of DOI:10.1021/ol062607b

(Chemical Equation Presented) A novel solid support 1 was synthesized to incorporate glyoxylic aldehyde functionality at the oligonucleotide 3′-terminus. 6-mer and 11-mer oligonucleotide sequences containing 3′-glyoxylic aldehyde functionality were prepar

Full text of DOI:10.1021/ol062607b

ORGANIC
LETTERS
2007
Vol. 9, No. 2
219-222
New Solid Support for the Synthesis of
-Oligonucleotide Conjugates through
3′
Glyoxylic Oxime Bond Formation
Nicolas Spinelli, Om Prakash Edupuganti, Eric Defrancq,* and Pascal Dumy
De´partement de Chimie Mole´culaire, UMR CNRS 5250, ICMG FR2607,
UniVersite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France
eric.defrancq@ujf-grenoble.fr
Received October 23, 2006
ABSTRACT
A novel solid support 1 was synthesized to incorporate glyoxylic aldehyde functionality at the oligonucleotide 3
′
-terminus. 6-mer and 11-mer
oligonucleotide sequences containing 3′-glyoxylic aldehyde functionality were prepared by using this support. These modified oligonucleotides
were coupled to reporters containing an aminooxy group to prepare oligonucleotide 3
The hydrolytic stability of a glyoxylic oxime linkage was also investigated.
′-conjugates through glyoxylic oxime bond formation.
Oligonucleotides (ODNs) have been extensively investigated
for selective inhibition of gene expression,1 DNA-based
vaccines,2 preparation of microarrays for diagnostic applica-
tions,3 and design of nanostructures4 with potential applica-
tions in electronic and/or photonic devices. However, certain
intrinsic oligonucleotide properties (such as stability to
degradation, cell uptake, and targeting, etc.) need to be further
optimized to completely realize the full potential of these
reagents. These intrinsic properties can be further improved
by modifying oligonucleotides with molecules such as
peptides,5 carbohydrates,6 lipids,7 metal complexes,8 and/or
fluorophores. Thus, design and development of synthetic
protocols for oligonucleotide conjugation has attracted
considerable research interest. The most common approach
used to prepare ODN conjugates involves separate prepara-
tion and purification of the oligonucleotide and the reporter
followed by their solution-phase coupling. This is achieved
by incorporating mutually reactive groups into each fragment,
and the process leads to the formation of stable chemical
linkages such as thioether,9 disulfide,10 oxime,11 and hydra-
zone.12 Recently, click chemistry13 has also been investigated
for ODN conjugation. Oxime linkages formed by the reaction
of an aldehyde with an aminooxy group remain the most
(9) (a) Harrison, J. G.; Balasubramanian, S. Nucleic Acids Res. 1998,
26, 3136-3145. (b) Arar, K.; Aubertin, A. M.; Roche, A. C.; Monsigny,
M.; Mayer, R. Bioconjugate Chem. 1995, 6, 573-577.
(10) (a) Antopolsky, M.; Azhayeva, E.; Tengvall, U.; Auriola, S.;
Jaaskelainen, I.; Ronkko, S.; Honkakoski, P.; Urtti, A.; Lonnberg, H.;
Azhayev, A. Bioconjugate Chem. 1999, 10, 598-606. (b) Maurel, F.;
Debart, F.; Cavelier, F.; Thierry, A. R.; Lebleu, B.; Vasseur, J. J.; Vives,
E. Bioorg. Med. Chem. Lett. 2005, 15, 5084-5087.
(1) Uhlmann, E.; Peyman, A. Chem. ReV. 1990, 90, 543-584.
(2) Klinman, D. M. Int. ReV. Immunol. 2006, 25, 135-154.
(3) Beaucage, S. L. Curr. Med. Chem. 2001, 8, 1213-1244.
(4) Gothelf, K. V.; LaBean, T. H. Org. Biomol. Chem. 2005, 3, 4023-
4037.
(5) (a) Tung, C. H.; Stein, S. Bioconjugate Chem. 2000, 11, 605-18.
(b) Gait, M. J. Cell Mol. Life Sci. 2003, 60, 844-853. (c) Venkatesan, N.;
Kim, B. H. Chem. ReV. 2006, 106, 3712-3761.
(6) Zatsepin, T. S.; Oretskaya, T. S. Chem. BiodiVersity 2004, 1, 1401-
1417.
(7) Gosse, C.; Boutorine, A.; Aujard, I.; Chami, M.; Kononov, A.; Cogne-
Laage, E.; Allemand, J.-F.; Li, J.; Jullien, L. J. Phys. Chem. B 2004, 108,
6485-6497.
(8) Grimm, G. N.; Boutorine, A. S.; Lincoln, P.; Norde´n, B.; He´le`ne, C.
ChemBioChem 2002, 3, 324-331.
(11) Zatsepin, T. S.; Stetsenko, D. A.; Gait, M. J.; Oretskaya, T. S.
Bioconjugate Chem. 2005, 16, 470-489.
(12) (a) Ollivier, N.; Olivier, C.; Gouyette, C.; Huynh-Dinh, T.; Gras-
Masse, H.; Melnyk, O. Tetrahedron Lett. 2002, 43, 997-999. (b) Raddatz,
S.; Mueller-Ibeler, J.; Kluge, J.; Wa¨ss, L.; Burdinski, G.; Havens, J. R.;
Onofrey, T. J.; Wang, D.; Schweitzer, M. Nucleic Acids Res. 2002, 30,
4793-4802.
(13) (a) Gierlich, J.; Burley, G. A.; Gramlich, P. M. E.; Hammond, D.
M.; Carell, T. Org. Lett. 2006, 8, 3639-3642. (b) Bouillon, C.; Meyer, A.;
Vidal, S.; Jochum, A.; Chevolot, Y.; Cloarec, J. P.; Praly, J. P.; Vasseur, J.
J.; Morvan, F. J. Org. Chem. 2006, 71, 4700-4702.
10.1021/ol062607b CCC: $37.00
© 2007 American Chemical Society
Published on Web 12/21/2006

widely used chemical linkage for ODN conjugation. This is
due to the fact that reactions leading to the formation of
oxime bonds are chemoselective and give high coupling
efficiency. Furthermore, these bonds are stable over a wide
pH range. Oxime bond formation has been extensively used
for the preparation of ODN conjugates with peptides,14
carbohydrates,15 and fluorescent probes.16 It has been shown
that ODN-glyoxylic aldehyde (R-ketoaldehyde)17 is more
stable to air oxidation and does not react with amino groups
during ligation. These glyoxylic aldehyde functionalities are
usually generated by periodate oxidation of a serine moiety
and have been extensively used in protein engineering.18
However, only a few methods have been reported in literature
that can be used to prepare ODN 5′-conjugates through
glyoxylic oxime bond formation.17,19 To the best of our
knowledge, there is no known method available for the
preparation of ODN 3′-conjugates by a similar method.
Recently, a method to prepare peptide-oligonucleotide
conjugates (POCs) through glyoxylic oxime linkage was
reported from our laboratory.19 The glyoxylic aldehyde linker
was incorporated at the 5′ extremity of ODN by using a novel
serine-containing phosphoramidite. The glyoxylic aldehyde
function was generated by the oxidation of the serine moiety,
and it was also shown that a glyoxylic oxime linkage is more
stable than an aldoxime linkage at acidic to neutral pH. It
was therefore decided to develop a protocol for the prepara-
tion of ODN 3′-conjugates through glyoxylic oxime bond
formation. This is of significant interest because 3′-modified
ODNs show greater resistance to nuclease activity compared
to 5′-analogues. Furthermore, 3′-conjugation keeps the 5′-
terminus free for 32P-labeling by kinase, which is a very
widespread technique used in molecular biology for DNA
gel analysis.
Figure 1. Solid support 1.
eptide motif is known to be a selective and powerful ligand
for Rvâ3 integrin receptors.20 The NLS peptide is a nuclear
localizing signal sequence with basic peptide APKKKRKVED
derived from the simian virus 40 antigen. The hydrolytic
stability of the 3′-glyoxylic oxime linkage was investigated
and compared to the stability of the 3′-aldoxime linkage.
The new long-chain alkyl amine-controlled pore glass
(LCAA-CPG) solid support 1 was prepared from com-
mercially available N-R-Fmoc-O-tBu-L-serine 2 in few
chemical steps (Scheme 1). 2 was converted to pentafluo-
Scheme 1. Preparation of Solid Support 1
We report, herein, a new and convenient procedure to
prepare oligonucleotides modified with glyoxylic aldehyde
at the 3′-terminus. This has been achieved by synthesizing
a novel solid support 1 for ODN synthesis and modification
(Figure 1). 6-mer and 11-mer oligonucleotide sequences
modified with 3′-glyoxylic aldehyde group were prepared
using this support. The efficiency of this procedure for 3′-
conjugation was investigated by coupling these ODN se-
quences to aminooxy-containing peptides (sequences con-
taining a RGD and NLS motif, respectively) and a fluorescein
derivative. The arginine-glycine-aspartic acid (RGD) trip-
t
rophenyl ester using pentafluorophenol/DCC. The Bu pro-
tecting group was removed using 100% trifluoroacetic acid
to obtain 3. This on N-acylation with 1-O-dimethoxytrityl-
6-amino-1-hexanol 4 gave compound 5. The procedure to
prepare protected amino linker 4 has been described earlier.21
It should be mentioned that â-hydroxy protected N-R-Fmoc-
O-tBu-L-serine 2 was used as the starting material instead
of â-hydroxy unprotected N-R-Fmoc-L-serine because the
latter compound showed significant side reactions on either
conversion to pentafluorenyl ester or acylation with 4 under
standard peptide coupling conditions. The synthon 5 was
anchored onto the solid support by using the “classical”
succinyl linker. The succinyl linker was attached at the
â-hydroxyl function of 5 via an esterification reaction with
succinic anhydride to obtain 6. The acid 6 was finally
attached to the solid support by reaction with the amino
(14) (a) Edupuganti, O. P.; Singh, Y.; Defrancq, E.; Dumy, P. Chem.-
Eur. J. 2004, 10, 5988-5995. (b) Zatsepin, T. S.; Stetsenko, D. A.;
Arzumanov, A. A.; Romanova, E. A.; Gait, M. J.; Oretskaya, T. S.
Bioconjugate Chem. 2002, 13, 822-830.
(15) (a) Singh, Y.; Renaudet, O.; Defrancq, E.; Dumy, P. Org. Lett. 2005,
7, 1359-1362. (b) Dey, S.; Sheppard, T. L. Org. Lett. 2001, 3, 3983-
3986.
(16) (a) Tre´visiol, E.; Renard, A.; Defrancq, E.; Lhomme, J. Nucleosides
Nucleotides Nucleic Acids 2000, 19, 1427-1439. (b) Salo, H.; Virta, P.;
Hakala, H.; Prakash, T. P.; Kawasaki, A. M.; Manoharan, M.; Lo¨nnberg,
H. Bioconjugate Chem. 1999, 10, 815-823.
(17) Far, S.; Gouyette, C.; Melnyk, O. Tetrahedron 2005, 61, 6138-
6142.
(18) (a) Liu, H.; Wang, L.; Brock, A.; Wong, C.-H.; Schultz, P. G. J.
Am. Chem. Soc. 2003, 125, 1702-1703. (b) Olivier, C.; Hot, D.; Huot, L.;
Ollivier, N.; El-Mahdi, O.; Gouyette, C.; Huynh-Dinh, T.; Gras-Masse, H.;
Lemoine, Y.; Melnyk, O. Bioconjugate Chem. 2003, 14, 430-439.
(19) Singh, Y.; Defrancq, E.; Dumy, P. J. Org. Chem. 2004, 69, 8544-
8546.
(20) Aumailley, M.; Gurrath, M.; Muller, G.; Calvete, J.; Timpl, R.;
Kessler, H. FEBS Lett. 1991, 291, 50-54.
(21) Avino, A.; Guimil Garcia, R.; Albericio, F.; Mann, M.; Wilm, M.;
Neubauer, G.; Eritja, R. Bioorg. Med. Chem. 1996, 4, 1649-1658.
220
Org. Lett., Vol. 9, No. 2, 2007

Scheme 2. Synthesis of Peptide-Oligonucleotide Conjugates Using Support 1 or Commercially Available Glyceryl CPG Supports
moiety present on the support using a standard procedure.22
Finally, unreacted amino groups on LCAA-CPG were capped
with acetic anhydride. The loading of the support was found
to 35 µmol/g as estimated from a dimethoxytrityl cation UV
assay. The advantage of using support 1 is that the Fmoc
group can be easily removed by an ammonia treatment which
is employed for nucleobase deprotection in standard DNA
synthesis protocols. Besides, the C6 linker should be suf-
ficient to minimize steric hindrance between the ODN and
the reporter.
The support 1 was first used to prepare 6-mer 9a and 11-
mer 9b oligonucleotide sequences (Scheme 2). ODN se-
quence 9a is a simple hexathymidylate. This was used to
optimize the automated synthesis on support 1, the oxidation
procedure, and the conjugation protocol. Oligonucleotide
elongation was performed by using the standard phosphora-
midite protocol on a 1 µmol scale. The ODNs were cleaved
from support and released into the solution by treatment with
ammonia.
The nucleobase deprotection and simultaneous removal
of the Fmoc group were accomplished by keeping the
ammonia solution at 55 °C for 16 h. The ODN sequences
so prepared were analyzed by RP-HPLC on a C18 column.
The HPLC profile showed the presence of a major ODN
product along with a minor amount of truncated sequences.
The 5′-DMT group was removed by treatment with 80%
aqueous acetic acid to obtain the oligonucleotide 7a contain-
ing the free serine at the 3′-end. The serine moiety was
successfully oxidized by using excess aqueous sodium
periodate to obtain ODN 3′-glyoxylic aldehyde 8a. The
oxidative cleavage was found to be clean, leading to the
exclusive formation of ODN 3′-glyoxylic aldehyde. As
reported earlier, 8a was found to be stable for months at
-20 °C.
The suitability of an ODN-glyoxylic aldehyde for 3′-
conjugation was investigated by synthesizing peptide oligo-
nucleotide conjugate 9a. The ODN-glyoxylic aldehyde 8a
was reacted with an aminooxy-containing RGD peptide23 in
0.1 M ammonium acetate buffer at room temperature. The
reaction was carefully monitored by RP-HPLC. The HPLC
profile showed the exclusive formation of conjugate 9a in
about 8 h (see the Supporting Information). The peptide-
ODN conjugate (POC) 9a containing the 3′-glyoxylic oxime
linkage was obtained in satisfactory yields after HPLC
purification. The 11-mer ODN-glyoxylic aldehyde 8b was
prepared by using a similar procedure. It should be mentioned
that the ODN sequence 8b is a heterooligonucleotide
containing different types of nucleotides, unlike 8a. This was
used to prepare peptide-ODN conjugate 9b as described
above. The efficiency of the method is further illustrated by
coupling the ODN-glyoxylic aldehyde 8b to a nuclear
localizing signal (NLS) peptide sequence functionalized with
an aminooxy function23 as well as to a fluorescein probe24
to afford the conjugates 10b and 11b, respectively.
The ODN derivatives and conjugates prepared herein were
characterized by ESIMS analysis. The observed molecular
(23) Forget, D.; Boturyn, D.; Defrancq, E.; Lhomme, J.; Dumy, P.
Chem.-Eur. J. 2001, 7, 3970-3984.
(24) Trevisiol, E.; Defrancq, E.; Lhomme, J.; Laayoun, A.; Cros, P.;
Tetrahedron 2000, 56, 6501-6510.
(22) Pon, R. T. Solid-Phase Supports for Oligonucleotide Synthesis;
Humana Press Inc.: Totowa, NJ, 1993; Vol. 20.
Org. Lett., Vol. 9, No. 2, 2007
221

weights were found to be in excellent agreement with
calculated values (Table 1).
Table 2. Percentage of Hydrolysis in Conjugates 9a and 12a at
Different pH (37 °C)
% hydrolysisa
Table 1. ESIMS Dataa
conjugate
9a
12a
7.0
∼9
∼10
∼13
compound
m/z calcd
m/z found
pH
4.0
<3
<3
<3
7.0
<3
<3
<5
9.0
<5
4.0
9.0
<6
<6
<6
7a
7b
8a
8b
9a
2027.4
3534.7
1996.4
3503.6
2654.7
4162.0
4755.4
4021.8
2541.6
2027.4
3533.3
1996.7
3502.6
2654.8
4160.5
4753.7
4020.2
2542.0
24 h
48 h
120 h
∼31
∼13
∼45
∼51
∼36
a Percentage of hydrolysis was estimated from the area under the peak
in the HPLC chromatogram (260 nm).
9b
10b
11b
12a
achieved by using a novel solid support 1 for ODN synthesis.
Support 1 was conveniently prepared from a commercially
available serine derivative in few steps. The support incor-
porates a masked glyoxylic aldehyde precursor at the 3′-
ODN terminus. Mild oxidation of this precursor leads to the
formation of ODN containing a 3′-glyoxylic aldehyde. ODN-
glyoxylic aldehydes undergo a chemoselective coupling
reaction with the aminooxy group to give ODN 3′-conjugates.
The glyoxylic oxime bonds showed higher stability than
aldoxime bonds at acidic to neutral pH but lower stability at
alkaline pH. The procedure developed herein can easily be
extended to the preparation of various other classes of
oligonucleotide conjugates.
a Analysis was carried out in the negative mode. CH3CN/H2O/Et3N (50:
50:2, v/v/v) was used as the eluent at a flow rate of 8 µL min-1
.
The hydrolytic stability of conjugate 9a containing the
glyoxylic-oxime bond was investigated. A control conjugate
12a containing an aldoxime linkage was also prepared for
comparison.23 Purified conjugates 9a and 12a were incubated
(10-5 M) in phosphate buffer solutions with pH values
adjusted to 4, 7, and 9. The percentage of hydrolysis of the
two linkages was estimated by RP-HPLC analysis after 24,
48, and 120 h. The data obtained are collected in Table 2,
and it clearly shows that the glyoxylic oxime conjugate is
more stable under acidic to neutral conditions. Only 5%
hydrolysis was observed in conjugate 9a (120 h, pH 4 and
7), whereas conjugate 12a showed 31% hydrolysis only in
24 h under similar conditions (pH 4). On the contrary,
conjugate 12a was found to be more stable under alkaline
conditions. 12a showed about 6% hydrolysis at pH 9 (120 h),
and conjugate 9a showed 36% hydrolysis under similar
conditions. The results obtained herein support our earlier
stability studies reported for ODN 5′-conjugates.19
Acknowledgment. This work was supported by the
Re´gion Rhone-Alpes, the “Centre National de la Recherche
Scientifique” (CNRS), and the “Institut Universitaire de
France”. We thank Dr Y. Singh for careful reading of this
manuscript.
Supporting Information Available: Experimental pro-
cedures, RP-HPLC profiles and ESIMS spectra, and Tm and
CD data. This material is available free of charge via the
Internet at http://pubs.acs.org.
Thus, a new and convenient procedure has been developed
for the synthesis of 3′-oligonucleotide conjugates through
the formation of glyoxylic oxime bonds. This has been
OL062607B
222
Org. Lett., Vol. 9, No. 2, 2007